The pivotal role of gonadotropin signaling in regulating gonadal development and functions has attracted much research attention in the past two decades. However, the precise physiological role of gonadotropin signaling is still largely unknown in fish. In this study, we have established both luteinizing hormone β-subunit (lhb) and luteinizing hormone receptor (lhr) knockout zebrafish lines by transcription activator-like effector nucleases. Intriguingly, both homozygous lhb and lhr mutant male fish are fertile. The fertilization rate, sperm motility and histological structure of the testis was not affected in both lhb and lhr mutant males. On the contrary, homozygous lhb mutant females are infertile while homozygous lhr mutant females are fertile. Folliculogenesis was not affected in both lhb and lhr mutants. But oocyte maturation and ovulation was disrupted in lhb mutant while only ovulation was affected in lhr mutant. Differential expression of genes in the ovary involved in steroidogenesis, oocyte maturation and ovulation was found between the lhb and lhr mutants. These data demonstrate the essential role of Lh signaling in oocyte maturation and ovulation, and support the notion that Lh acts through Fshr in the absence of Lhr. Moreover, the defects of lhb mutant could be partially restored by administration of hCG. This in vivo evidence in the present study demonstrates, for the first time in any vertebrate species, that Lh signaling is indispensable in female reproduction but not in male reproduction. Lh signaling is demonstrated to control oocyte maturation and ovulation in the ovary.